An Immunochemical Approach to Detect the Quorum Sensing-Regulated Virulence Factor 2-Heptyl-4-Quinoline N-Oxide (HQNO) Produced by Pseudomonas aeruginosa Clinical Isolates

ABSTRACT Understanding quorum sensing (QS) and its role in the development of pathogenesis may provide new avenues for diagnosing, surveillance, and treatment of infectious diseases. For this purpose, the availability of reliable and efficient analytical diagnostic tools suitable to specifically detect and quantify these essential QS small molecules and QS regulated virulence factors is crucial. Here, we reported the development and evaluation of antibodies and an enzyme-linked immunosorbent assay (ELISA) for HQNO (2-heptyl-4-quinoline N-oxide), a QS product of the PqsR system, which has been found to act as a major virulence factor that interferes with the growth of other microorganisms. Despite the nonimmunogenic character of HQNO, the antibodies produced showed high avidity and the microplate-based ELISA developed could detect HQNO in the low nM range. Hence, a limit of detection (LOD) of 0.60 ± 0.13 nM had been reached in Müeller Hinton (MH) broth, which was below previously reported levels using sophisticated equipment based on liquid chromatography coupled to mass spectrometry. The HQNO profile of release of different Pseudomonas aeruginosa clinical isolates analyzed using this ELISA showed significant differences depending on whether the clinical isolates belonged to patients with acute or chronic infections. These data point to the possibility of using HQNO as a specific biomarker to diagnose P. aeruginosa infections and for patient surveillance. Considering the role of HQNO in inhibiting the growth of coinfecting bacteria, the present ELISA will allow the investigation of these complex bacterial interactions underlying infections. IMPORTANCE Bacteria use quorum sensing (QS) as a communication mechanism that releases small signaling molecules which allow synchronizing a series of activities involved in the pathogenesis, such as the biosynthesis of virulence factors or the regulation of growth of other bacterial species. HQNO is a metabolite of the Pseudomonas aeruginosa-specific QS signaling molecule PQS (Pseudomonas quinolone signal). In this work, the development of highly specific antibodies and an immunochemical diagnostic technology (ELISA) for the detection and quantification of HQNO was reported. The ELISA allowed profiling of the release of HQNO by clinical bacterial isolates, showing its potential value for diagnosing and surveillance of P. aeruginosa infections. Moreover, the antibodies and the ELISA reported here may contribute to the knowledge of other underlying conditions related to the pathology, such as the role of the interactions with other bacteria of a particular microbiota environment.


MATERIALS AND INSTRUMENTS:
Chemistry. The  Immunochemistry. Chemicals and biochemicals were obtained from Aldrich Chemical Co.
(Milwaukee, WI, USA) and from Sigma Chemical Co. (St. Louis, MO, USA). The HHQ (2-heptyl-4quinolone) hapten used in this study was prepared following a similar synthetic procedure as that described by Reen et al.[41] and conjugated to BSA (HHQ-BSA, hapten density 13). The stock solutions of the alkylquinolones (HHQ, PQS, HQNO) used as standards were prepared in DMSO at 10 mM and stored at -20ºC, then transferred to 4ºC prior to their use. Purification of the bioconjugates was carried out in ÄKTA Prime Plus using 2 HiTrap desalting columns both from GE Healthcare (Chicago, IL, USA) or either by dialysis using Spectra/Por membranes from Spectrumlabs (Piraeus, Greece, EU) with molecular weight cut-off of 12-14 kDa. The matrixassisted laser desorption ionization time-of-flight mass spectrometer (MALDI-TOF-MS) was a Bruker Autoflex III Smartbeam spectrometer (Billerica, Massachusetts). Hapten densities of the bioconjugates were calculated by MALDI-TOF-MS by comparing the molecular weight recorded on the MALDI spectra of the native proteins to that of the HQNO-BSA bioconjugates. For this purpose, the bioconjugates were mixed with the freshly prepared matrix ((trans-3,5-dimethoxy-4-hydroxycinnamic acid, 10 mg mL -1 in 70:30 ACN/H2O, 0.1% HCOOH) following the ''sandwich'' sample preparation method. The bioconjugate aliquot was diluted ½ using CAN (0.2% HCOOH).

Polyclonal antisera (PAb).
Three female New Zealand white rabbits weighing 1-2 kg were immunized with HQNO-KLH following established protocols in the research group.
Immunizations were carried out in the animal facility of the Research and Development Center (CID) of the Spanish Research Council (CSIC) Registration Number: B9900083, employing approved procedures that avoid unnecessary treatments and minimize suffering of the animals.
The protocol used in accordance with the institutional guidelines under a license from the local government (DAAM 7463) and approved by the Institutional Animal Care and Use Committee at the CID-CSIC. The antisera (As) obtained were named As388, As389 and As390. The animals were exsanguinated after 6 immunizations, and the final blood was collected in vacutainer tubes provided with a serum separation gel. Antisera were obtained by centrifugation at 4 °C for 10 min at 10 000 rpm, then stored at −80 °C in the presence of preservative 0,02% sodium azide.
The antibody titer was assessed during the immunization process through non-competitive indirect ELISA. Microtiter plates were coated with a fixed concentration of HQNO-BSA conjugate (1 mg mL −1 ) and the avidity of the produced antibodies was measured by preparing serial dilutions of the corresponding As.

Non-competitive indirect two-dimensional titration experiments. Titration experiments in
non-competitive indirect ELISA format were performed in order to establish the best conditions of As dilution and concentration of coating antigen (CA) for the competitive quantification assays. With this aim, the dilutions of antisera (1/1000 to 1/64000, 100 μL/well) were added to Microtiter plates, which were previously coated with BSA competitors (5 μg mL -1 to 5 ng mL -1 , coating buffer, 100 μL/well) and washed with PBST (4 x 300 μL/well), and the binding measured following the protocol described in section 2.4.2. The combination of As dilution and CA concentration producing around 0.7-1.2 units of absorbance was selected.   The parameters improving the features of the assay were assessed separately and in conjunction.
ELISA EVALUATION: Figure S2. Matrix effect of the MH broth undiluted and diluted 2, 5, 10 and 20 times with PBST on the As389/ HHQ-BSA ELISA. The calibration curves were run using the conditions established for the assay in PBS-6.5. Modification of the assay conditions allowed achieving similar immunoassay features as when the assay was run in buffer (see Figure 2). The results shown are the average and standard deviations of analysis made on two different days measured by duplicates each day.